HIV currently affects 35 million people worldwide. Infection occurs by the interaction of the envelope glycoprotein gp120 on the surface of the HIV particle with CD4 and one of two chemokine receptors, CXCR4 or CCR5, on host cells, which promotes viral entry by gp41-mediated fusion of viral and host cell membranes. CCR5-tropic viruses are primarily responsible for initial infection and transmission of the disease, while CXCR4-tropic viruses are correlated with later stage progression to full-blown AIDS. Consequently, since 1981, significant efforts have been deployed to develop potent small molecule, peptide and protein entry inhibitors of HIV coreceptors, and a CCR5 entry inhibitor, Maraviroc, was approved in 2007. The emergence of CXCR4 tropic virus in ~50% percent of infected individuals and its correlation with more rapid decline in CD4+ cell counts and faster progression to the symptomatic stage of AIDS has also encouraged development of CXCR4 inhibitors. Structural information on how gp120 interacts with CXCR4 to enter cells would be highly valued to advance such endeavors, as well as to provide a better understanding of viral entry, tropism and resistance. While structures of gp120 have been solved with other soluble proteins involved in entry of HIV into cells, structural understanding of its interaction with full length chemokine coreceptors is still lacking. This is due to the challenge of determining structures involving membrane proteins, particularly with protein ligands like chemokines and gp120. Recently, we solved the structure of the first complex of CXCR4 with a protein ligand, the viral chemokine vMIP-II. Structures of CXCR4 with the HIV protein ligand, gp120, represent an obvious extension of these studies. Our long term goal is to obtain a structural understanding of CCR5 and CXCR4 interactions with HIV gp120, thus enabling rational design of highly efficient HIV entry inhibitors with reduced susceptibility to development of resistance. The objective of the present proposal is therefore to generate stable complexes of full length CXCR4 with gp120 as a prelude to future structural studies. Our central hypothesis is that gp120 interaction with the co-receptors structurally mimics that of chemokines and is mediated by at least two distinct epitopes. We will focus on binding determinants centered on the tropism determining gp120 V3 loop. Furthermore, we will use a computationally-guided disulfide trap strategy developed in the course of determining the CXCR4:vMIP-II structure to identify disulfides that spontaneously form between non-native cysteines introduced into gp120 and CXCR4 in order to make stable CXCR4:gp120 complexes suitable for structural studies. In the process of doing so, we will determine distance restraints that can be used to model complexes of the interaction, and develop a platform for understanding the tolerance of CXCR4 to gp120 sequence diversity in the context of HIV resistance and tropism. The proposal is innovative in both its challenging objectives and its novel disulfide trap methodology, and it is significant because it will advance the understanding of HIV entry, tropism, resistance and therapeutic design.